Adipocytes promote ovarian cancer chemoresistance

Abstract

Ovarian cancer (OvCa), while accounting for only 3% of all women's cancer, is the fifth leading cause of cancer death among women. One of the most significant obstacles to successful OvCa treatment is chemoresistance. The current lack of understanding of the driving mechanisms underlying chemoresistance hinders the development of effective therapeutics against this obstacle. Adipocytes are key components of the OvCa microenvironment and have been shown to be involved in OvCa cell proliferation, however, little is known about their impact on OvCa chemoresistance. In the current study, we found that adipocytes, of both subcutaneous and visceral origin, secrete factors that enhance the resistance of OvCa cells against chemotherapeutic drugs by activating the Akt pathway. Importantly, we have demonstrated that secreted lipids mediate adipocyte-induced chemoresistance. Through a comprehensive lipidomic analysis, we have identified this chemo-protective lipid mediator as arachidonic acid (AA). AA acts on OvCa cells directly, not through its downstream derivatives such as prostaglandins, to activate Akt and inhibit cisplatin-induced apoptosis. Taken together, our study has identified adipocytes and their secreted AA as important mediators of OvCa chemoresistance. Strategies that block the production of AA from adipocytes or block its anti-apoptotic function may potentially inhibit chemoresistance in OvCa patients.

Figure 1

Adipocytes enhance ovarian cancer cell chemoresistance. (A) Human OvCa cells OVCAR5, CAOV3 and SKOV3 were incubated with adipocyte CM and different concentrations of cisplatin. Cell viability was analyzed 72 hours later. (B) Ovarian cancer cells were incubated with adipocyte CM together with different concentrations of paclitaxel or doxorubicin. (C) OVCAR5 cells were incubated with CM from visceral adipocytes (visAdi_CM), together with cisplatin. (D) Apoptosis marker cleaved PARP was analyzed in lysates from OVCAR5 cells treated with cisplatin and subcutaneous (subAdi_CM) or visceral adipocyte CM (visAdi_CM). *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 2

The Akt pathway mediates the anti-apoptotic activity of adipocytes. Akt was activated by both subcutaneous adipocyte CM (subAdi_CM) (A) and visceral/omental adipocyte CM (OmAdi_CM) (B) in human OVCAR5 cells and by the CM from mouse adipocytes differentiated from 3T3-L1 cells in ID8 mouse OvCa cells (C). Adipocyte-induced Akt activation in OVCAR5 cells was inhibited by LY294002 dose-dependently (D). LY294002 abolished the anti-apoptotic effect of adipocyte CM, as shown by increased level of cleaved caspase-3 and cleaved PARP (E).

Figure 3

The anti-apoptotic activity of adipocyte CM resides predominantly in the <3 kDa fraction. The <3 kDa fraction of adipocyte CM exerted similar effects as unfractionated adipocyte CM, i.e., reducing the apoptosis in OVCAR5 cells in the presence of cisplatin, whereas the >3 kDa fraction did not. (A) Representative dot plot of flow cytometry data after Annexin V staining. (B) Fold change in the percentage of Annexin V-positive apoptotic cells in different treatment groups (n = 3). (C) PARP immunoblot with two independently collected batches of CM.

Figure 4

Lipids mediate the anti-apoptotic function of adipocytes. (A) Lipids in the <3 kDa fraction of adipocyte CM extracted with methyl formate (M/F) inhibited cisplatin-induced apoptosis in OVCAR5 cells to a similar extent as did the unfractionated CM, as shown by PARP immunoblot. (B) Lipids in adipocyte CM extracted with MTBE also reduced cisplatin-induced apoptosis, as shown by Annexin V staining. Two independently collected batches of adipocyte CM (Batch 1 and 2) were analyzed. *p < 0.05.

Figure 5

Prostaglandins do not mediate adipocyte-induced chemoresistance. Prostaglandin D₂ (PGD₂) (A), PGE₂ (B) and PGF_2α (C) at different concentrations were applied on OVCAR5 cells in the presence of 1 μM cisplatin. Cell viability was analyzed after 72 hours. PGD₂ applied at 2 μg/mL caused extensive cytotoxicity. (D) Prostaglandin levels were analyzed in the CM collected from adipocytes treated with COX1 and COX2 inhibitor celecoxib. (E) Blockade of prostaglandin synthesis with celecoxib did not compromise the anti-apoptotic function of adipocytes. (F) Adipocyte CM treatment reduced cisplatin-induced apoptosis even when PGE₂ receptors were blocked on OVCAR5 cells with specific inhibitors.

Figure 6

Arachidonic acid induces chemoresistance in ovarian cancer cells. (A) OVCAR5 cells were treated with 1 μM cisplatin and different concentrations of arachidonic acid (AA). Cell viability was analyzed 72 hours later. (B) AA inhibited cisplatin-induced apoptosis, as demonstrated by PARP immunoblotting. (C) Akt was activated in OVCAR5 cells by AA. **p < 0.01, as compared to control cells treated with cisplatin.

Figure 7

Adipocyte CM and arachidonic acid increase chemoresistance in both chemo-sensitive and -resistant ovarian cancer cells. Chemosensitve PEO1 cells (A) and chemoresistant PEO4 cells (B) were incubated with adipocyte CM and different concentrations of cisplatin. PEO1 cells (C) and PEO4 cells (D) were also treated with 5 μM or 20 μM cisplatin, respectively, and different concentrations of arachidonic acid (AA). Cell viability was analyzed 72 hours later. *p < 0.05, **p < 0.01 and ***p < 0.001.